Signal recognition particle polypeptides and polynucleotides

ABSTRACT

This invention relates to a novel bacterial ribonucleoprotein complex and the component parts thereof. More specifically, this invention relates to SRP isolated from  Staphylococcus aureus  and the use of SRP or components thereof in screens for the identification of antimicrobial compounds and to the use of such compounds in therapy.

FIELD OF THE INVENTION

[0001] This invention relates to newly identified polynucleotides,polypeptides encoded by certain of these polynucleotides, molecularcomplexes of RNAs and polypeptides, the uses of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides and recombinant host cells transformed with thepolynucleotides. The invention relates particularly to suchpolynucleotides and polypeptides from Staphylococci, especially S.aureus. This invention also relates to inhibiting the biosynthesis,assembly or action of such polynucleotides and/or polypeptides and tothe use of such inhibitors in therapy.

BACKGROUND OF THE INVENTION

[0002] This invention relates to a novel bacterial ribonucleoproteincomplex and the component parts thereof. More specifically, thisinvention relates to SRP, particularly SRP from Staphylococcus aureus.and the use of SRP or components thereof in screens for theidentification of antimicrobial compounds and to the use of suchcompounds in therapy.

[0003] The Staphylococci make up a medically important genera ofmicrobes. They are known to produce two types of disease, invasive andtoxigenic. Invasive infections are characterized generally by abscessformation effecting both skin surfaces and deep tissues. S. aureus isthe second leading cause of bacteremia in cancer patients.Osteomyelitis, septic arthritis, septic thrombophlebitis and acutebacterial endocarditis are also relatively common. There are at leastthree clinical conditions resulting from the toxigenic properties ofStaphylococci. The manifestation of these diseases result from theactions of exotoxins as opposed to tissue invasion and bacteremia. Theseconditions include: Staphylococcal food poisoning, scalded skin syndromeand toxic shock syndrome

[0004] The frequency of Staphylococcus aureus infections has, risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Staphylococcus aureus strains which are resistant tosome or all of the standard antibiotics. This has created a demand forboth new anti-microbial agents and diagnostic tests for this organism

[0005] While certain Staphylococcal proteins associated withpathogenicity have been identified, e.g., coagulase, hemolysins,leucocidins and exo- and enterotoxins, additional targets are alwaysuseful because it is appreciated that the target of a antimicrobialscreen can often bias the outcome. Thus, new targets allow for thediscovery of new classes of antimicrobials.

BRIEF DESCRIPTION OF THE INVENTION

[0006] This invention provides a novel ribonucleoprotein complex,particularly such complex from Staphylococcus aureus, and the separatelyisolated RNA and protein components thereof.

[0007] In accordance with another aspect of the present invention, thereare provided polynucleotides (DNA or RNA) which encode the protein andRNA components of such a complex.

[0008] In particular the invention provides polynucleotides having theDNA sequences given herein.

[0009] The invention also relates to novel oligonucleotides derived fromthe sequences given herein which can act, for example, as antisenseinhibitors of the expression of the RNA or protein components. Theoligonucleotides or fragments or derivatives thereof can be used todirectly inhibit binding or other biological activity or indirectlyinhibit activity by interference with RNA protein complex formation. Theprotein and the RNA components, either separately or in a complex, arealso useful as targets in screens designed to identify antimicrobialcompounds.

[0010] It is an object of the invention to provide polypeptides thathave been identified as novel SRP polypeptides by homology between knownamino acid sequences, such as B. subtilis Ffh and E. coli Ffh.

[0011] It is a further object of the invention to providepolynucleotides that encode SRP polypeptides, particularlypolynucleotides that encode the polypeptide herein designated Ffh, aswell as polynucleotides that are transcribed into SRP RNA, particularlypolynucleotides that encode the RNA herein designated Ffs.

[0012] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding Ffh polypeptides comprisingthe sequence set out in Table 1 [SEQ ID NO:1] which includes a fulllength gene, or a variant thereof.

[0013] In another particularly preferred embodiment of the inventionthere is a novel Ffh protein from Staphylococcus aureus comprising theamino acid sequence of Table 1 [SEQ ID NO:2], or a variant thereof.

[0014] In accordance with another aspect of the invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the Staphylococcus aureus WCUH 29 strain contained in thedeposited strain.

[0015] A further aspect of the invention there are provided isolatednucleic acid molecules encoding SRP, particularly Staphylococcus aureusSRP, including mRNAs, cDNAs, genomic DNAs and structural RNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful variants thereof,and compositions comprising the same.

[0016] In accordance with another aspect of the invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of SRP and polypeptides encoded thereby.

[0017] Another aspect of the invention there are provided novelpolypeptides of Staphylococcus aureus referred to herein as Ffh as wellas biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

[0018] Among the particularly preferred embodiments of the invention arevariants of Ffh polypeptide encoded by naturally occurring alleles ofthe ffh gene.

[0019] In a preferred embodiment of the invention there are providedmethods for producing the aforementioned Ffh polypeptides.

[0020] In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

[0021] In accordance with certain preferred embodiments of theinvention, there are provided products, compositions and methods forassessing SRP expression, treating disease, for example, disease, suchas, infections of the upper respiratory tract (e.g. otitis media,bacterial tracheitis, acute epiglottitis, thyroiditis), lowerrespiratory (e.g., empyema, lung abscess), cardiac (e.g., infectiveendocarditis), gastrointestinal (e.g., secretory diarrhoea, splenicabscess, retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (eg. blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptaland orbital cellulitis, darcryocystitis), kidney and urinary tract(e.g., epididymitis, intrarenal and perinephric absces, toxic shocksyndrome), skin (e.g., impetigo, folliculitis, cutaneous abscesses,cellulitis, wound infection, bacterial myositis) bone and joint (e.g.,septic arthritis, osteomyelitis), assaying genetic variation, andadministering a Ffh polypeptide or polynucleotide to an organism toraise an immunological response against a bacteria, especially aStaphylococcus aureus bacteria.

[0022] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided polynucleotides thathybridize to SRP polynucleotide sequences, particularly under stringentconditions.

[0023] In certain preferred embodiments of the invention there areprovided antibodies against Ffh polypeptides.

[0024] In other embodiments of the invention there are provided methodsfor identifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

[0025] In accordance with yet another aspect of the invention, there areprovided SRP agonists and antagonists, preferably bacteriostatic orbacteriocidal agonists and antagonists.

[0026] In a further aspect of the invention there are providedcompositions comprising a SRP polynucleotide or a SRP polypeptide foradministration to a cell or to a multicellular organism.

[0027] Various changes and modifications within the spirit and scope, ofthe disclosed, invention will become readily apparent to those skilledin the art from reading the following descriptions and from reading theother parts of the present disclosure.

[0028] Glossary:

[0029] The following definitions are provided to facilitateunderstanding of certain terms used frequently herein. Certain otherdefinitions are provided elsewhere herein.

[0030] “Host cell” is a cell which has been transformed or transfected,or is capable of transformation or transfection by an exogenouspolynucleotide sequence.

[0031] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). As an illustration, by a polynucleotide having anucleotide sequence having at least, for example, 95% “identity” to areference nucleotide sequence of SEQ ID NO:1 it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence of SEQ ID NO:1. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% of the nucleotides in the referencesequence may be deleted or substituted with another nucleotide, or anumber of nucleotides up to 5% of the total nucleotides in the referencesequence may be inserted into the reference sequence. These mutations ofthe reference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

[0032] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

[0033] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

[0034] “Polypeptide(s)” refers to any peptide or protein comprising twoor more amino acids joined to each other by peptide bonds or modifiedpeptide bonds. “Polypeptide(s)” refers to both short chains, commonlyreferred to as peptides, oligopeptides and oligomers and to longerchains generally referred to as proteins. Polypeptides may contain aminoacids other than the 20 gene encoded amino acids. “Polypeptide(s)”include those modified either by natural processes, such as processingand other post-translational modifications, but also by chemicalmodification techniques. Such modifications are well described in basictexts and in more detailed monographs, as well as in a voluminousresearch literature, and they are well known to those of skill in theart. It will be appreciated that the same type of modification may bepresent in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Modifications can occur anywhere in a polypeptide,including the peptide backbone, the amino acid side-chains, and theamino or carboxyl termini. Modifications include, for example,acetylation, acylation. ADP-ribosylation, anidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, metlylation, myristoylationoxidation, proteolytic processing, phosphorylation, prenylation,racemization, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation andADP-ribosylation, selencylation, sulfation, transfer-RNA mediatedaddition of amino acids to proteins, such as arginylation, andubiquitination. See, for instance, PROTEINS-STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth. Etzymol. 182:626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

[0035] “Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynucleotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The ribonucleoprotein, Signal Recognition Particle (hereinafterreferred to as SRP), consists of a protein component known as Ffh and aribonucleic acid component encoded by the ffs gene. SRP plays a key rolein the targetting of nascent intrinsic cytoplasmic membrane proteins tothe membrane and in the targetting of some soluble proteins to theperiplasm in Gram-negative bacteria or to extra-cellular locations inGram-positive bacteria. SRP is known to interact with the FtsY protein,the product of the ftsY gene, in eubacteria and also with hydrophobicregions of pre-secretory proteins and with hydrophobic regions ofintrinsic membrane proteins. The interaction between SRP and FtsYresults in reciprocal stimulation of the intrinsic guanosinetriphosphate (GTP) hydrolytic activity of each protein. Loss ofbiological activity of either Ffh protein or FtsY protein isincompatible with bacterial cell survival.

[0037] The invention relates to novel SRP polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novel Ffh ofStaphylococcus aureus, which is related by amino acid sequence homologyto Ffh polypeptide set forth in SEQ ID NO:2. The invention relatesespecially to Ffh having the nucleotide and amino acid sequences set outin Table 1, SEQ ID NO:1 and SEQ ID NO:2 respectively, and to the ffhnucleotide sequences of the DNA in the deposited strain and amino acidsequences encoded thereby. The invention also relates to the SRP RNAcomponent, particularly in its protein binding form, and sequences fromwhich such component is transcribed.

[0038] SRP RNA Component:

[0039] Phylogenetic comparisons readily allow secondary structuremodeling and the identification of a minimum consensus structure.Several ribonucleosides within the ffs sequence of eubacteria which havean important role in functioning of the SRP have been identified.

[0040] SRP Protein Component:

[0041] The precise functional role of the protein involves recognitionof and binding to ffs RNA, GTP, FtsY and hydrophobic regions ofpre-secretory proteins and with hydrophobic regions of intrinsicmembrane proteins. The interaction between SRP and FtsY results inreciprocal stimulation of the intrinsic guanosin(triphosphate (GTP)hydrolytic activity of each protein.

[0042] The full length sequence encoding the intact SRP proteincomponent can be obtained by probing a genomic library by for example insitu colony hybridization detailed in Maniatis et al. (infra) using aprobe(s) generated based on the sequences given in SEQ ID NOS: 1 and/or2. TABLE 1 SRP Polynucleotide and Polypeptide Sequences (A) Sequencesfrom Staphylococcus aureus ffh polynucleotide sequence [SEQ ID NO:1].5′-ATGGCATTTGAAGGCTTATCAGAACGCCTGCAAGCGACGATGCAAAAAATGCGTGGTAAGGGTAAACTTACTGAAGCTGATATAAAGATAATGATGCGTGAAGTAAGATTAGCGTTATTTGAGGCTGACGTAAACTTTAAAGTGGTAAAAGAATTTATTAAAACAGTATCAGAACGCGCATTAGGTTCCGATGTAATGCAATCATTAACACCAGGGCAACAAGTTATTAAAATAGTTCAAGATGAATTAACGAAGTTGATGGGTGGAGAAAATACATCGATTAATATGTCAAATAAACCACCTACTGTTGTTATGATGGTTGGTTTACAAGGTGCTGGTAAAACAACAACTGCAGGTAAATTAGCATTATTGATGCGTAAAAAATACAACAAAAAACCTATGTTAGTTGCAGCAGATATTTATCGTCCAGCAGCGATAAATCAATTACAAACAGTAGGGAAACAAATTGATATTCCTGTATACAGTGAAGGAGATCAAGTAAAGCCACAACAAATTGTAACTAATGCATTAAAACATGCTAAAGAAGAACATTTAGACTTTGTAATCATTGATACAGCAGGTCGATTACACATCGATGAAGCATTGATGAACGAATTAAAAGAAGTAAAAGAAATTGCTAAACCAAACGAAATTATGTTAGTTGTCGATTCAATGACGGGTCAAGATGCTGTCAATGTTGCAGAATCTTTTGACGATCAACTTGATGTCACAGGTGTTACCTTAACTAAATTAGATGGTGATACCCGTGGTGGTGCAGCTTTATCTATTCGT-3′ (B) Ffh polypeptidesequence deduced from the polynucleotide sequence in this table [SEQ IDNO:2]. NH-MAFEGLSERLQATMQKMRGKGKLTEADIKIMMREVRLALFEADVNFKVVKEFIKTVSERALGSDVMQSLTPGQQVIKIVQDELTKLMGGENTSINMSNKPPTVVMMVGLQGAGKTTTAGKLALLMRKKYNKKPMLVAALIYRPAAINQLQTVGKQIDIPVYSEGDQVKPQQIVTNALKHAKEEHLDFVIIDTAGRLHIDEALMNELKEVKEIAKPNEIMLVVDSMTGQDAVNVAESFDDQLDVTGVTLTKLDGDTRGGAALSIR-COOH (C) Polynucleotide sequence embodiments [SEQ IDNO:1]. X−(R₁)_(n) ATGGCATTTGAAGGCTTATCAGAACGCCTGCAAGCGACGATGCAAAAAATGCGTGGTAAGGGTAAACTTACTGAAGCTGATATAAAGATAATGATGCGTGAAGTAAGATTAGCGTTATTTGAGGCTGACGTAAACTTTAAAGTGGTAAAAGAATTTATTAAAACAGTATCAGAACGCGCATTAGGTTCCGATGTAATGCAATCATTAACACCAGGGCAACAAGTTATTAAAATAGTTCAAGATGAATTAACGAAGTTGATGGGTGGAGAAAATACATCGATTAATATGTCAAATAAACCACCTACTGTTGTTATGATGGTTGGTTTACAAGGTGCTGGTAAAACAACAACTGCAGGTAAATTAGCATTATTGATGCGTAAAAAATACAACAAAAAACCTATGTTAGTTGCAGCAGATATTTATCGTCCAGCAGCGATAAATCAATTACAAACAGTAGGGAAACAAATTGATATTCCTGTATACAGTGAAGGAGATCAAGTAAAGCCACAACAAATTGTAACTAATGCATTAAAACATGCTAAAGAAGAACATTTAGACTTTGTAATCATTGATACAGCAGGTCGATTACACATCGATGAAGCATTGATGAACGAATTAAAAGAAGTAAAAGAAATTGCTAAACCAAACGAAATTATGTTAGTTGTCGATTCAATGACGGGTCAAGATGCTGTCAATGTTGCAGAATCTTTTGACGATCAACTTGATGTCACAGGTGTTACCTTAACTAAATTAGATGGTGATACCCGTGGTGGTGCAGCTTTATCTATTCGT −(R₂)_(n)−Y (D) Polypeptidesequence embodiments [SEQ ID NO:2]. +TL,X−(R₁)_(n−)MAFEGLSERLQATMQKMRGKGKLTEADIKIMMREVRLALFEADVNFKVVKEFIKTVSERALGSDVMQSLTPGQQVIKIVQDELTKLMGGENTSINMSNKPPTVVMMVGLQGAGKTTTAGKLALLMRKKYNKKPMLVAADIYRPAAINQLQTVGKQIDIPVYSEGDQVKPQQIVTNALKHAKEEHLDFVIIDTAGRLHIDEALMNELKEVKEIAXPNEIMLVVDSMTGQDAVNVASSFDDQLDVTGVTLTR LDGDTRGGAALSIR−(R₂)_(n−Y) (E) Sequences from Staphylococcus aureus SRP RNA gene ffs[SEQ ID NO:3]. 5′-AACAATGCCGTTTCAATATAATATTTCAAAACATCTTGCAAATGAATTTAAATTTACCGACTTCTCAAGACGTCGTATAAAGTAAACAATGATATAAATGATTTATACTTGCAATTAACTATTNAAATATAGTAATATATATCTTTCCGTGCTAGGTGGGGAGGTAGCGGTTCCCTGTACTCGAAATCCGCTTTATGCGAGGCTTAATTCCTTTGTTGAGGCCGTATTTTTGCGAAGTCTGCCCAAAGCACGTAGTGTTTGAAGATTTCGGTCCT-3′ (F) Polynucleotide sequence embodiments[SEQ ID NO:3]. X−(R₁)_(n)AACAATGCCGTTTCAATATAATATTTCAAAACATCTTGCAAATGAATTTAAATTTACCGACTTCTCAAGACGTCGTATAAAGTAAACAATGATATAAATGATTTATACTTGCAATTAACTATTNAAATATAGTAATATATATCTTTCCGTGCTAGGTGGGGAGGTAGCGGTTCCCTGTACTCGAAATCCGCTTTATGCGAGGCTTAATTCCTTTGTTGAGGCCGTATTTTTGCGAAGTCTGCCCAAAGCACGTAGTGTTTGAAGATTTCGGTCCT−(R₂)_(n−)Y

[0043] Polypeptides of the Invention:

[0044] The polypeptides of the invention include the polypeptide ofTable 1 [SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of Ffh, and also those which have at least 70% identity to thepolypeptide of Table 1 [SEQ ID NO:2] or the relevant portion, preferablyat least 80% identity to the polypeptide of Table 1 [SEQ ID NO:2], andmore preferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of Table 1 [SEQ ID NO:2] and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide of Table 1 [SEQ ID NO:2] and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

[0045] The invention also includes polypeptides of the formula set forthin Table 1 (D) [SEQ ID NO:2] wherein, at the amino terminus, X ishydrogen, and at the carboxyl terminus, Y is hydrogen or a metal, R₁ andR₂ is any amino acid residue, and n is an integer between 1 and 1000 orzero. Any stretch of amino acid residues denoted by either R group,where R is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer.

[0046] A fragment is a variant polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned polypeptides. As with Ffh polypeptides fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion, a single larger polypeptide.

[0047] Preferred fragments include, for example, truncation polypeptideshaving a portion of the amino acid sequence of Table 1 [SEQ ID NO:2], orof variants thereof, such as a continuous series of residues thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus. Degradation forms of the polypeptides ofthe invention in a host cell, particularly a Staphylococcus aureus, arealso preferred. Further preferred are fragments characterized bystructural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

[0048] Also preferred are biologically active fragments which are thosefragments that mediate activities of Ffh, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Also included are those fragments that are antigenic orimmunogenic in an animal, especially in a human. Particularly preferredare fragments comprising receptors or domains of enzymes that confer afunction essential for viability of Staphylococcus aureus or the abilityto initiate, or maintain cause disease in an individual, particularly ahuman.

[0049] Variants that are fragments of the polypeptides of the inventionmay be employed for producing the corresponding full-length polypeptideby peptide synthesis; therefore, these variants may be employed asintermediates for producing the full-length polypeptides of theinvention.

[0050] Polynucleotides of the Invention:

[0051] Another aspect of the invention relates to isolatedpolynucleotides, including the full length gene, that encode the Ffhpolypeptide having a deduced amino acid sequence of Table 1 [SEQ IDNO:2] and polynucleotides closely related thereto and variants thereof.

[0052] Using the information provided herein, such as a polynucleotidesequence set out in SEQ ID NOS. 1 or 3, a polynucleotide of theinvention encoding SRP polypeptide or RNA (such as that transcribed fromSEQ ID NO:3) may be obtained using standard cloning and screeningmethods, such as those for cloning and sequencing chromosomal DNAfragments from bacteria using Staphylococcus aureus WCUH 29 cells asstarting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as asequence given in SEQ ID NOS:1 or 3, typically a library of clones ofchromosomal DNA of Staphylococcus aureus WCUH 29 in E. coli or someother suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing stringent conditions. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Conveniently, such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Illustrative of theinvention, the polynucleotides set out in Table 1 [SEQ ID NO:1] werediscovered in a DNA library derived from Staphylococcus aureus WCUH 29.

[0053] Certain DNA sequences set out in Table 1 [SEQ ID NO:1] containsan open reading frame encoding a protein having about the number ofamino acid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known in the art.

[0054] Ffh of the invention is structurally related to other proteins ofthe Ffh family, as shown by the results of sequencing the DNA encodingFfh of the deposited strain. The protein exhibits greatest homology toB. subtilis protein among known proteins. Ffh of Table 1 [SEQ ID NO:2]has signficant identity and similarity over its entire length with theamino acid sequence of B. subtilis Ffh polypeptide.

[0055] The invention provides a polynucleotide sequence identical overits entire length to the coding sequence in Table 1 [SEQ ID NO:1]. Alsoprovided by the invention is the coding sequence for the maturepolypeptide or a fragment thereof, by itself as well as the codingsequence for the mature polypeptide or a fragment in reading frame withother coding sequence, such as those encoding a leader or secretorysequence, a pre-, or pro- or prepro-protein sequence. The polynucleotidemay also contain non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences, termination signals, ribosome binding sites,sequences that stabilize mRNA, introns, polyadenylation signals, andadditional coding sequence which encode additional amino acids. Forexample, a marker sequence that facilitates purification of the fusedpolypeptide can be encoded. In certain embodiments of the invention, themarker sequence is a hexa-histidine peptide, as provided in the pQEvector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.Sci., USA 86: 821-824 (1989), or an HA tag (Wilson et al., Cell 37: 767(1984). Polynucleotides of the invention also include, but are notlimited to, polynucleotides comprising a structural gene and itsnaturally associated sequences that control gene expression.

[0056] A preferred embodiment of the invention is a polynucleotide setforth in SEQ ID NO:1 of Table 1 which encodes the Ffh polypeptide.

[0057] The invention also includes polynucleotides of the formula setforth in Table 1 (C)[SEQ ID NO:1 and (F)[SEQ ID NO:3] wherein, at the 5′end of the molecule, X is hydrogen, and at the 3′ end of the molecule, Yis hydrogen or a metal, R₁ and R₂ is any nucleic acid residue, and n isan integer between 1 and 3000 or zero. Any stretch of nucleic acidresidues denoted by either R group, where R is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer. Apreferred embodiment for the sequence set forth in Table 1 (F) [SEQ IDNO:3] has R₁ or R₂ being between 1 and 10 or 1 and 20, and especiallybeing 1 or 3. The invention also provides RNA transcribed from suchpolynucleotides, particularly RNAs that bind an Ffh polypeptide.

[0058] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus Ffh havingthe amino acid sequence set out in Table 1 [SEQ ID NO:2]. The term alsoencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or an insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

[0059] The invention further relates to variants of the polynucleotidesdescribed herein that encode for variants of the polypeptide having thededuced amino acid sequence of Table 1 [SEQ ID NO:2]. Variants that arefragments of the polynucleotides of the invention may be used tosynthesize full-length polynucleotides of the invention.

[0060] In addition to the standard A, G, C, T/U representations fornucleic acid bases, the term “N” is also used. “N” means that any of thefour DNA or RNA bases may appear at such a designated position in theDNA or RNA sequence, except that, in preferred embodiments, N can not bea base that when taken in combination with adjacent nucleotidepositions, when read in the correct reading frame, would have the effectof generating a premature termination codon in such reading frame.

[0061] Further particularly preferred embodiments are polynucleotidesencoding Ffh variants, that have the amino acid sequence of Ffhpolypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of SRP.

[0062] Further preferred embodiments of the invention arepolynucleotides that are at least 50%, 60% or 70% identical over theirentire length to a polynucleotide encoding Ffh polypeptide having anamino acid sequence set out in Table 1 [SEQ ID NO:2], andpolynucleotides that are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical over its entire length to apolynucleotide encoding Ffh polypeptide of the deposited strain andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

[0063] Preferred embodiments of the invention are polynucleotides thatare at least 50%, 60% or 70% identical over their entire length to anFfh polynucleotide having a nucleotide sequence set out in SEQ ID NO:3and polynucleotides that are complementary to such polynucleotides.Alternatively, most highly preferred are polynucleotides that comprise aregion that is at least 80% identical over its entire length to an Ffhpolynucleotide of the deposited strain and polynucleotides complementarythereto. In this regard, polynucleotides at least 90% identical overtheir entire length to the same are particularly preferred, and amongthese particularly preferred polynucleotides, those with at least 95%are especially preferred. Furthermore, those with at least 97% arehighly preferred among those with at least 95%, and among these thosewith at least 98% and at least 99% are particularly highly preferred,with at least 99% being the more preferred. It is especially preferredthat these polynucleotides be RNAs, especially RNAs that bind Ffhpolypeptides.

[0064] Preferred embodiments are polynucleotides that encodepolypeptides that retain substantially the same biological function oractivity as the mature polypeptide encoded by the DNA of Table 1 [SEQ IDNO:1 ] or as the SRP RNA component transcribed by the DNA of SEQ IDNO:3.

[0065] The invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the herein above-described polynucleotides. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization will occur only if there is at least 95%and preferably at least 97% identity between the sequences. An exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mMtrisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution. 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the hybridization support in0.1×SSC at about 65° C. Hybridization and wash conditions are well knownand exemplified in Sambrook, et at., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularlyChapter 11 therein.

[0066] The invention also provides a polynucleotide consistingessentially of a polynucleotide sequence obtainable by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO:1 or SEQ ID NO:3 under stringenthybridization conditions with a probe having the sequence of saidpolynucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3respectively or a fragment thereof; and isolating said DNA sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers described elsewhere herein.

[0067] As discussed herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding SRP and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the SRP gene. Such probes generally wall comprise at least15 bases. Preferably, such probes will have at least 30 bases and mayhave at least 50 bases. Particularly preferred probes will have at least30 bases and will have 50 bases or less.

[0068] Polynucleotides of the invention that are oligonucleotidesderived from the sequences of SEQ ID NOS:1 and/or 2 and/or 3 may be usedin the processes herein as described, but preferably for PCR, todetermine whether or not the polynucleotides identified herein in wholeor in part are transcribed in bacteria in infected tissue. It isrecognized that such sequences will also have utility in diagnosis ofthe stage of infection and type of infection the pathogen has attained.

[0069] The invention also provides polynucleotides that may encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

[0070] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0071] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

[0072] Further Description and Definitions:

[0073] The coding region of the SRP gene may be isolated, for example,by screening using a deposit containing a Staphylococcus aureus WCUH 29strain which has been deposited with the National Collections ofIndustrial and Manne Bacteria Ltd. (herein “NCIMB”), 23 St. MacharDrive, Aberdeen AB2 IRY, Scotland on Sep. 11, 1995 and assigned NCIMBDeposit No. 40771. It was referred to as Staphylococcus aureus WCUH29 ondeposit The Staphylococcus aureus strain deposit is referred to hereinas “the deposited strain” or as “the DNA of the deposited strain.”

[0074] The deposited strain contains the full length SRP gene. Thesequence of the polynucleotides contained in the deposited strain, aswell as the amino acid sequence of the polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

[0075] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

[0076] A license may be required to make, use or sell the depositedstrain, and compounds derived therefrom, and no such license is herebygranted.

[0077] The nucleotide sequences disclosed herein can also be obtained bysynthetic chemical techniques known in the art or can be obtained fromS. aureus WCUH 29 by probing a DNA preparation with probes constructedfrom the particular sequences disclosed herein. Alternatively,oligonucleotides derived from a disclosed sequence can act as PCRprimers in a process of PCR-based cloning of the sequence from abacterial genomic source. It is recognized that such sequences will alsohave utility in diagnosis of the type of infection the pathogen hasattained.

[0078] A polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, geromic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the polypeptide may beidentical to the coding sequence shown or may be a different codingsequence which coding sequence, as a result of the redundancy ordegeneracy of the genetic code, encoding the same polypeptide.

[0079] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a ploynucleotide which includes additional codingand/or non-coding sequence.

[0080] The present invention therefore includes polynucleotides, whereinthe coding sequence for the mature polypeptide may be fused in the samereading frame to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains.

[0081] Thus, for example, the polynucleotide of the present inventionmay encode for a mature protein, or for a protein having a prosequenceor for a protein having both a prosequence and a presequence (leadersequence). Further, the amino acid sequences provided herein show amethionine residue at the NH₂-terminus. It is appreciated, however, thatduring post-translational modification of the peptide, this residue maybe deleted. Accordingly, this invention contemplates the use of both themethionine-containing and the methionineless amino terminal variants ofeach protein disclosed herein.

[0082] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence at either the 5′ or3′ terminus of the gene which allows for purification of the polypeptideof the present invention. The marker sequence may be a hexa-histidinetag supplied by the pQE series of vectors (supplied commercially byQuiagen Inc.) to provide for purification of the polypeptide fused tothe marker in the case of a bacterial host. Alternatively the maltosebinding protein (MBP) fusion system may be employed. In this system thegene of interest is fused the male gene encoding the MBP (supplied byNew England BioLabs). The fusion product is purified in a one stepprocedure based on the MBP affinity for maltose. A pre-engineered Xacleavage site allows for efficient removal of the MBP component from thegene product of interest.

[0083] In order to facilitate understanding of the following examplecertain frequently occurring methods and/or terms will be described.

[0084] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0085] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on an agarose gel to isolate the desired fragment. Sizeseparation of the cleaved fragments is generally performed using a 1%percent agarose gel.

[0086] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0087] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., supra., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units to T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0088] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity

[0089] A “replicon” is any genetic element (e.g., plasmid, chromosome,virus) that functions as an autonomous unit of DNA replication in vivo;i.e., capable of replication under its own control.

[0090] A “vector” is a replicon, such as a plasmid, phage, or cosmid, towhich another DNA segment may be attached so as to bring about thereplication of the attached segment.

[0091] A “double-stranded DNA molecule” refers to the polymeric form ofdeoxyribonicleotides (bases adenine, guanine, thymine, or cytosine) in adouble-stranded helix, both relaxed and supercoiled. This term refersonly to the primary and secondary structure of the molecule, arid doesnot limit it to any particular tertiary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments, viruses, plasmids, and chromosomes. In discussingthe structure of particular double-stranded DNA molecules, sequences maybe described herein according to the normal convention of giving onlythe sequence in the 5′ to 3′ direction along the nontranscribed strandof DNA (i.e., the strand having the sequence homologous to the mRNA).

[0092] A DNA “coding sequence of” or a “nucleotide sequence encoding” aparticular protein, is a DNA sequence which is transcribed andtranslated into a polypeptide when placed under the control ofappropriate regulatory sequences.

[0093] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bound at the 3′ terminus bya translation start codon (e.g., ATG) of a coding sequence and extendsupstream (5′ direction) to include the minimum number of bases orelements necessary to initiate transcription at levels detectable abovebackground. Within the promoter sequence will be found a transcriptioninitiation site (conveniently defined by mapping with nuclease S1), aswell as protein binding domains (consensus sequences) responsible forthe binding of RNA polymerase. Eukaryotic promoters will often, but notalways, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoterscontain the −10 and −35 consensus sequences.

[0094] DNA “control sequences” refers collectively to promotersequences, ribosome binding sites, polyadenylation signals,transcription termination sequences, upstream regulatory domains,enhancers, and the like, which collectively provide for the expression(i.e., the transcription and translation) of a coding sequence in a hostcell.

[0095] A control sequence “directs the expression” of a coding sequencein a cell when RNA polymerase will bind the promoter sequence andtranscribe the coding sequence into mRNA, which is then translated intothe polypeptide encoded by the coding sequence.

[0096] A “host cell” is a cell which has been transformed ortransfected, or is capable of transformation or transfection by anexogenous DNA sequence.

[0097] A cell has been “transformed” by exogenous DNA when suchexogenous DNA has been introduced inside the cell membrane Exogenous DNAmay or may not be integrated (covalently linked) into chromosomal DNAmaking up the genome of the cell In prokaryotes and yeasts, for example,the exogenous DNA may be maintained on an episomal element, such as aplasmid. With respect to eukaryotic cells, a stably transformed ortransfected cell is one in which the exogenous DNA has become integratedinto the chromosome so that it is inherited by daughter cells throughchromosome replication. This stability is demonstrated by the ability ofthe eukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cell containing the exogenous DNA.

[0098] A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

[0099] A “heterologous” region of a DNA construct is an identifiablesegment of DNA within or attached to another DNA molecule that is notfound in association with the other molecule in nature.

[0100] Preparation of the SRP Protein Component:

[0101] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0102] In accordance with yet a further aspect of the present invention,there is therefore provided a process for producing the polypeptide ofthe invention by recombinant techniques by expressing a polynucleotideencoding said polypeptide in a host and recovering the expressedproduct. Alternatively, the polypeptides of the invention can besynthetically produced by conventional peptide synthesizers.

[0103] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a cosmid, a phage, etc. Theengineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression, and will be apparent to the ordinarilyskilled artisan.

[0104] Suitable expression vectors include chromosomal, nonchromosomaland synthetic DNA sequences, e.g., bacterial plasmids; phage DNA;baculoviris; Beast plasmids; vectors derived from combinations ofplasmids and phase DNA. However, any other vector may be used as long asit is replicable and viable in the host.

[0105] For recombinant production, host cells can be geneticallyengineered to incorporate expression systems or portions thereof orpolynucleotides of the invention. Introcduction of a polynucleotide intothe host cell can be effected by methods described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989), such as, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction and infection.

[0106] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, enterococci E. coli,streptomyces and Bacillis subtilis cells: fungal cells, such as yeastcells and Aspergillus cells; insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3,BHK, 293 and Bowes melanoma cells; and plant cells.

[0107] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids. The expression system constructs maycontain control regions that regulate as well as engender expression.Generally, any system or vector suitable to maintain, propagate orexpress polynucleotides and/or to express a polypeptide in a host may beused for expression in this regard. The appropriate DNA sequence may beinserted into the expression system by any of a variety of well-knownand routine techniques, such as, for example, those set forth inSambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, (supra).

[0108] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart.

[0109] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in eukaryotic or prokaryotic cells or their viruses. Theexpression vector may also contains a ribosome binding site fortranslation initiation and/or a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression.

[0110] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0111] The gene can be placed under the control of a promoter, ribosomebinding site (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired protein is transcribed into RNA in thehost cell transformed by a vector containing this expressionconstruction. The coding sequence may or may not contain a signalpeptide or leader sequence. The polypeptides of the present inventioncan be expressed using, for example, the E. coli tac promoter or theprotein A gene (spa) promoter and signal sequence. Leader sequences canbe removed by the bacterial host in post-translational processing. See,e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397. Promoter regionscan be selected from any desired gene using CAT (chloramphenicoltransferase) vectors or other vectors with selectable markers. Twoappropriate vectors are PKK232-8 and PCM7. Particular named bacterialpromoters include lacI, lacZ, T3, T7, gpt, lambda P_(R), P_(L) and trp.Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40. LTRs from retrovirus, and mouse metallothionein-I.Selection of the appropriate vector and promoter is well within thelevel of ordinary skill in the art.

[0112] In addition to control sequences, it may be desirable to addregulatory sequences which allow for regulation of the expression of theprotein sequences relative to the growth of the host cell. Regulatorysequences are known to those of skill in the art, and examples includethose which cause the expression of a gene to be turned on or off inresponse to a chemical or physical stimulus, including the presence of aregulatory compound. Other types of regulatory elements may also bepresent in the vector, for example, enhancer sequences.

[0113] An expression vector is constructed so that the particular codingsequence is located in the vector with the appropriate regulatorysequences, the positioning and orientation of the coding sequence withrespect to the control sequences being such that the coding sequence istranscribed under the “control” of the control sequences (i.e., RNApolymerase which binds to the DNA molecule at the control sequencestranscribes the coding sequence). Modification of the coding sequencesmay be desirable to achieve this end. For example, in some cases it maybe necessary to modify the sequence so that it may be attached to thecontrol sequences with the appropriate orientation; i.e., to maintainthe reading frame. The control sequences and other regulatory sequencesmay be ligated to the coding sequence prior to insertion into a vector,such as the cloning vectors described above. Alternatively, the codingsequence can be cloned directly into an expression vector which alreadycontains the control sequences and an appropriate restriction site.

[0114] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably, a leader sequencecapable of directing secretion of translated protein into theperiplasmic space or extracellular medium. Optionally, the heterologoussequence can encode a fusion protein including an N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct.

[0115] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0116] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vector, and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pET-3 vectors (Stratagene),pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK. pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Suatagene):ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:pBlueBacIII (Invitrogen), pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL, (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0117] Examples of recombinant DNA vectors for cloning and host cellswhich they can transform include the bacteriophage λ (E. coli), pBR322(E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), a baculovirus insect cell system, YCp19(Saccharommyces). See, generally, “DNA Cloning”: Vols. I & II, Glover etal. ed. IRL Press Oxford (1985) (1987) and; T. Maniatis et al.(“Molecular Cloning” Cold Spring Harbor Laboratory (1982).

[0118] In some cases, it may be desirable to add sequences which causethe secretion of the polypeptide from the host organism, with subsequentcleavage of the secretory signal.

[0119] Polypeptides can be expressed in host cells under the control ofappropriate promoters. Cell-free translation systems can also beemployed to produce such proteins using RNAs derived from the DNAconstructs of the present invention. Appropriate cloning and expressionvectors for use with prokaryotic and eukaryotic hosts are described bySambrook, et al., Molecular Cloning: A Laboratory Manual, SecondEdition. Cold Spring Harbor, N.Y., (1989), the disclosure of which ishereby incorporated by reference.

[0120] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0121] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0122] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well known to those skilled in the art.

[0123] Depending on the expression system and host selected, thepolypeptide of the present invention may be produced by growing hostcells transformed by an expression vector described above underconditions whereby the polypeptide of interest is expressed. Thepolypeptide is then isolated from the host cells and purified. If theexpression system secretes the polypeptide into growth media, thepolypeptide can be purified directly from the media If the polypeptideis not secreted, it is isolated from cell lysates or recovered from thecell membrane fractions. Where the polypeptide is localized to the cellsurface. whole cells or isolated membranes can be used as an assayablesource of the desired gene product. Polypeptide expressed in bacterialhosts such as E. coli may require isolation from inclusion bodies andrefolding. Where the mature protein has a very hydrophobic region whichleads to an insoluble product of overexpression, it may be desirable toexpress a truncated protein in which the hydrophobic region has beendeleted. The selection of the appropriate growth conditions and recoverymethods are within the skill of the art.

[0124] The polypeptide can be recovered and purified from recombinantcell cultures by methods including ammonium sulphate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0125] Depending upon the host employed in a recombinant productionprocedure, the polypeptides of the present invention may be glycosylatedor may be non-glycosylated. Polypeptides of the invention may alsoinclude an initial methionine amino acid residue.

[0126] Preparation of the SRP RNA Component:

[0127] The SRP RNA molecules are prepared by run-off in vitrotranscription using T7 RNA polymerase as according to standardconditions usually as recommended by the supplier, e.g., Promega. Theplasmid is linearized with an appropriate restriction enzyme generatinga linear dsDNA comprising the full length gene encoding the SRP RNA. TheRNA is purified either from a preparative denaturing acrylamide gel oris precipitated prior to use in in vitro cleavage assays. The RNA canalso be prepared by automated synthesis.

[0128] Antagonists and Agonists—Assays and Molecules

[0129] This invention provides a method of screening drugs to identifythose which interfere with the RNA portion, the protein portion and/orthe intact RNA/protein complex of the SRP described herein, which methodcomprises measuring the interference of the activity of the proteinand/or RNA by a test drug. For example since the RNA portion selectedhas a binding activity for Ffh polypeptide, after suitable purificationand formulation the activity of the RNA can be followed by its abilityto convert its natural or synthetic RNA substrates By incorporatingdifferent chemically synthesized test compounds or natural products intosuch an assay of enzymatic activity one is able to detect thoseadditives which compete with the natural or synthetic substrate orotherwise inhibit enzymatic activity.

[0130] Polypeptides of the invention may also be used to assess thebinding of small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics. See, e.g., Coliganet al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

[0131] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof SRP polypeptides or polynucleotides, particularly those compoundsthat are bacteriostatic and/or bacteriocidal. The method of screeningmay involve high-throughput techniques. For example, to screen foragonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising Ffh polypeptide and a labeledsubstrate or ligand of such polypeptide is incubated in the absence orthe presence of a candidate molecule that may be a SRP agonist orantagonist. The ability of the candidate molecule to agonize orantagonize the Ffh polypeptide is reflected in decreased binding of thelabeled ligand or decreased production of product from such substrate.Molecules that bind gratuitously, i.e., without inducing the effects ofFfh polypeptide are most likely to be good antagonists. Molecules thatbind well and increase the rate of product production from substrate areagonists. Detection of the rate or level of production of product fromsubstrate may be enhanced by using a reporter system. Reporter systemsthat may be useful in this regard include but are not limited tocolorimetric labeled substrate converted into product, a reporter genethat is responsive to changes in SRP polynucleotide or polypeptideactivity, and binding assays known in the art.

[0132] Another example of an assay for SRP antagonists is a competitiveassay that combines SRP and a potential antagonist with SRP-bindingmolecules, recombinant SRP binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. SRP can be labeled, such as byradioactivity or a colorimetric compound, such that the number of SRPmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

[0133] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing SRP-induced activities, thereby preventing the action of SRP byexcluding SRP from binding.

[0134] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988). for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of SRP.

[0135] Each of the DNA sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

[0136] The invention also provides the use of the polypeptide,polynucleotide or inhibitor of the invention to interfere with theinitial physical interaction between a pathogen and mammalian hostresponsible for sequelae of infection. In particular the molecules ofthe invention may be used: in the prevention of adhesion of bacteria, inparticular gram positive bacteria, to mammalian extracellular matrixproteins on in-dwelling devices or to extracellular matrix proteins inwounds; to block SRP protein-mediated mammalian cell invasion by, forexample, initiating phosphorylation of mammalian tyrosine kinases(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterialadhesion between mammalian extracellular matrix proteins and bacterialFfh proteins that mediate tissue damage and; to block the normalprogression of pathogenesis in infections initiated other than by theimplantation of in-dwelling devices or by other surgical techniques

[0137] The antagonists and agonists of the invention may be employed,for instance, to inhibit and treat disease, such as, infections of theupper respiratory tract (e.g., otitis media, bacterial tracheitis,acute, epiglottitis, thyroiditis), tower respiratory (e.g., empyema,lung abscess), cardiac (e.g., infective endocarditis). gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

[0138] HTP Screening Strategies:

[0139] HTS strategy will be to measure the hydrolysis of GTP bymeasuring the accumulation of phosphate.

[0140] Diagnostic Assays

[0141] This invention is also related to the use of the SRPpolynucleotides of the invention for use as diagnostic reagents.Detection of SRP in a eukaryote, particularly a mammal, and especially ahuman, will provide a diagnostic method for diagnosis of a disease.Eukaryotes (herein also “individual(s)”), particularly mammals, andespecially humans, infected with an organism comprising the SRP gene maybe detected at the nucleic acid level by a variety of techniques.

[0142] Nucleic acids for diagnosis may be obtained from an infectedindividual's cells and tissues, such as bone, blood, muscle, cartilage,and skin. Genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR or other amplification techniqueprior to analysis. RNA or cDNA may also be used in the same ways. Usingamplification, characterization of the species and strain of prokaryotepresent in an individual, may be made by an analysis of the genotype ofthe prokaryote gene. Deletions and insertions can be detected by achange in size of the amplified product in comparison to the genotype ofa reference sequence. Point mutations can be identified by hybridizingamplified DNA to labeled SRP polynucleotide sequences. Perfectly matchedsequences can be distinguished from mismatched duplexes by RNasedigestion or by differences in melting temperatures. DNA sequencedifferences may also be detected by alterations in the electrophoreticmobility of the DNA fragments in gels, with or without denaturingagents, or by direct DNA sequencing. See, e.g. Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase and S1 protectionor a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl.Acad. Sci. USA, 85:4397-4401 (1985)

[0143] Cells carrying mutations or polymorphisms in the gene of theinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to a nucleicacid encoding SRP can be used to identify and analyze mutations.Examples of representative primers are shown in the Examples. Theinvention further provides these primers with 1, 2, 3 or 4 nucleotidesremoved from the 5′ and/or the 3′ end. These primers may be used for,among other things, amplifying SRP DNA isolated from a sample derivedfrom an individual. The primers may be used to amplify the gene isolatedfrom an infected individual such that the gene may then be subject tovarious techniques for elucidation of the DNA sequence. In this way,mutations in the DNA sequence may be detected and used to diagnoseinfection and to serotype and/or classify the infectious agent.

[0144] The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStaphylococcus aureus, and most preferably disease, such as, infectionsof the upper respiratory tract (e.g., otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.,empyema, lung abscess), cardiac (e.g., infective endocarditis),gastrointestinal (e.g., secretory diarrhoea, splenic absces,retroperitoneal abscess), CNS (e.g., cerebral abscess), eye (e.g.,blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.,epididymitis, intrarenal and pennephric absces, toxic shock syndrome),skin (e.g., impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g., septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of Table 1 [SEQ ID NO: 1]. Increased or decreasedexpression of SRP polynucleotide can be measured using any on of themethods well known in the art for the quantitation of polynucleotides,such as, for example, amplification, PCR, RT-PCR, SRProtection, Northernblotting and other hybridization methods.

[0145] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of Ffh protein compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a Ffhprotein, in a sample derived from a host are well-known to those ofskill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.

[0146] Antibodies

[0147] The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

[0148] The Fab fragment may also be prepared from its parent monoclonalantibody by enzyme treatment, for example using papain to cleave the Fabportion from the Fc portion.

[0149] Antibodies generated against the polypeptides of the inventioncan be obtained by administering the polypeptides or epitope-bearingfragments, analogues or cells to an animal, preferably a nonhuman, usingroutine protocols. The antibody so obtained will then bind thepolypeptides itself. In this manner, even a sequence encoding only afragment of the polypeptides can be used to generate antibodies bindingthe whole native polypeptides. Such antibodies can then be used toisolate the polypeptide from tissue expressing that polypeptide. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985. in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

[0150] The hybridomas are screened to select a cell line with highbinding affinity and favorable cross reaction with other staphylococcalspecies using one or more of the original polypeptide and/or the fusionprotein. The selected cell line is cultured to obtain the desired Mab.

[0151] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies

[0152] Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Ffh or from naive libraries (McCafferty, J.et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

[0153] The antibody should be screened again for high affinity to thepolypeptide and/or fusion protein.

[0154] As mentioned above, a fragment of the final antibody may beprepared.

[0155] The antibody may be either intact antibody of M_(r) approx150,000 or a derivative of it, for example a Fab fragment or a Fvfragment as described in Skerra, A and Pluckthun, A., Science240:1038-1040 (1988). If two antigen binding domains are present eachdomain may be directed against a different epitope—termed ‘bispecific’antibodies.

[0156] If two antigen binding domains are present each domain may bedirected against a different epitope—termed ‘bispecific’ antibodies.

[0157] In particular derivatives which are slightly longer or slightlyshorter than the native protein or polypeptide fragment of the presentinvention may be used. In addition, polypeptides in which one or more ofthe amino acid residues are modified may be used. Such peptides may, forexample, be prepared by substitution, addition, or rearrangement ofamino acids or by chemical modification thereof. All such substitutionsand modifications are generally well known to those skilled in the artof peptide chemistry.

[0158] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides to purify the polypeptidesby affinity chromatography.

[0159] Thus, among others, antibodies against Ffh-polypeptide may beemployed to treat infections, particularly bacterial infections andespecially disease, such as, infections of the upper respiratory tract(e.g., otitis media, bacterial tracheitis, acute epiglottitis,thyroiditis), lower respiratory (e.g., empyema, lung abscess), cardiac(e.g., infective endocarditis), gastrointestinal (e.g., secretorydiarrhoea, splenic absces, retroperitoneal abscess), CNS (e.g., cerebralabscess), eye (e.g., blepharitis, conjunctivitis, keratitis,endophthalmitis, preseptal and orbital cellulitis, darcryocystitis),kidney and urinary tract (e.g., epididymitis, intrarenal and perinephricabsces, toxic shock syndrome), skin (e.g., impetigo, folliculitis,cutaneous abscesses, cellulitis, wound infection, bacterial myositis)bone and joint (e.g., septic arthritis, osteomyelitis).

[0160] Preferably the antibody is prepared by expression of a DNApolymer encoding said antibody in an appropriate expression system suchas described above for the expression of polypeptides of the invention.The choice of vector for the expression system will be determined inpart by the host, which may be a prokaryotic cell, such as E. coli(preferably strain B) or Streptomyces sp. or a eukaryotic cell, such asa mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary,filamentous or unicellular fungi or insect cell. The host may also be atransgenic animal or a transgenic plant (for example, as described inHiatt, A. et al., Nature 340:76-78(1989). Suitable vectors includeplasmids, bacteriophages, cosmids and recombinant viruses, derived from,for example, baculoviruses and vaccinia.

[0161] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

[0162] The polypeptide, such as an antigenically or immunologicallyequivalent derivative or a fusion protein thereof is used as an antigento immunize a mouse or other animal such as a rat or chicken. The fusionprotein may provide stability to the polypeptide. The antigen may beassociated, for example by conjugation, with an immunogenic carrierprotein for example bovine serum albumin (BSA) or keyhole limpethaemocyanin (KLH). Alternatively a multiple antigenic peptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide thereof may be sufficientlyantigenic to improve immunogenicity so as to obviate the use of acarrier.

[0163] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etat., (1991) Biotechnology 9, 266-273. The humanized monoclonal antibody,or its fragment having binding activity, form a particular aspect ofthis invention.

[0164] The modification need not be restricted to one of “humanization”;other primate sequences (for example Newman, R. et a., Biotechnology 10:1455-1460 (1992)) may also be used.

[0165] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. 1989: 264,16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, 1986:83,9551), encapsulationof DNA in various forms of liposomes (Kaneda et al., Science1989:243,375), particle bombardment (Tang et al., Nature 1992, 356:152,Eisenbraun et al., DNA Cell Biol 1993, 12:791) and in vivo infectionusing cloned retroviral vectors (Seeger et al., PNAS USA 1984:81,5849).

[0166] Vaccines

[0167] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal whichcomprises inoculating the individual with SRP, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Staphylococcus aureus infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector to direct expression of SRP, or afragment or a variant thereof, for expressing SRP, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not. One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise. Such nucleic acid vector may comprise DNA, RNA,a modified nucleic acid, or a DNA/RNA hybrid.

[0168] A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a SRP or protein coded therefrom, whereinthe composition comprises a recombinant SRP or protein coded therefromcomprising DNA which codes for and expresses an antigen of said SRP orprotein coded therefrom. The immunological response may be usedtherapeutically or prophylactically and may take the form of antibodyimmunity or cellular immunity such as that arising from CTL or CD4+Tcells.

[0169] A Ffh polypeptide or a fragment thereof may be fused withco-protein which may not by itself produce antibodies, but is capable ofstabilizing the first protein and producing a fused protein which willhave immunogenic and protective properties. Thus fused recombinantprotein, preferably further comprises an antigenic co-protein, such aslipoprotein D from Hemophilus influenzae. Glutathione-S transferase(GST) or beta-galactosidase, relatively large co-proteins whichsolubilize the protein and facilitate production and purificationthereof. Moreover, the co-protein may act as an adjuvant in the sense ofproviding a generalized stimulation of the immune system. The co-proteinmay be attached to either the amino or carboxy terminus of the firstprotein.

[0170] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

[0171] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStaphylococcus aureus infection, in mammals, particularly humans

[0172] The polypeptide may be used as an antigen for vaccination of ahost to produce specific antibodies which protect against invasion ofbacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

[0173] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation insotonic with the bodily fluid, preferably the blood, ofthe individual; and aqueous and non-aqueous sterile suspensions whichmay include suspending agents or thickening agents. The formulations maybe presented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0174] While the invention has been described with reference to certainFfh protein, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

[0175] Compositions, Kits and Administration

[0176] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or their agonists orantagonists. The polypeptides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration. Theinvention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention

[0177] Polypeptides and other compounds of the invention may be employedalone or in conjunction with other compounds, such as therapeuticcompounds.

[0178] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0179] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0180] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0181] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/Kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0182] In-dwelling devices include surgical implants, prosthetic devicesand catheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

[0183] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStaphylococcus aureus wound infections.

[0184] Many orthopaedic surgeons consider that humans with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteremia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It maytherefore be possible to extend the use of the active agent as areplacement for prophylactic antibiotics in this situation.

[0185] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins exposed in wound tissueand for prophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

[0186] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 1 μg/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0187] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is preferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0188] Each reference disclosed herein is incorporated by referenceherein in its entirety. Any patent application to which this applicationclaims priority is also incorporated by reference herein in itsentirety.

EXAMPLES

[0189] The present invention is further described by the followingexamples. The examples are provided solely to illustrate the inventionby reference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

[0190] All examples were carried out using standard techniques, whichare well known and routine to those of skill in the art, except whereotherwise described in detail. Routine molecular biology techniques ofthe following examples (can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor laboratory Press, ColdSpring Harbor, N.Y. (1989).

[0191] All parts or amounts set out in the following examples are byweight, unless otherwise specified.

[0192] Unless otherwise stated size separation of fragments in theexamples below was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) and numerous otherreferences such as, for instance, by Goeddel et al., Nucleic Acids Res.8: 4057 (1980).

Example 1 Strain Selection, Library Production and Sequencing

[0193] The polynucleotide having the DNA sequence given in SEQ ID NO: 1was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO: 1 Libraries may beprepared by routine methods, for example:

[0194] Methods 1 and 2 Below.

[0195] Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

[0196] Method 1

[0197] Total cellular DNA is mechanically sheared by passage through aneedle in order to size-fractionate according to standard procedures.DNA fragments of up to 11 kbp in size are rendered blunt by treatmentwith exonuclease and DNA polymerase, and EcoRI linkers added. Fragmentsare ligated into the vector Lambda ZapII that has been cut with EcoRI,the library packaged by standard procedures and E. coli infected withthe packaged library. The library is amplified by standard procedures.

[0198] Method 2

[0199] Total cellular DNA is partially hydrolyzed with a one or acombination of restriction enzymes appropriate to generate a series offragments for cloning into library vectors (e.g. RsaI, PaII, AluI,Bsh235I), and such fragments are size-fractionated according to standardprocedures. EcoRI linkers are ligated to the DNA and the fragments thenligated into the vector Lambda ZapII that have been cut with EcoRI, thelibrary packaged by standard procedures, and E. coli infected with thepackaged library The library

What is claimed is:
 1. An isolated polynucleotide segment, comprising anucleic acid sequence, wherein the nucleic acid sequence encodes apolypeptide comprising SEQ ID NO:2.
 2. A vector comprising the isolatedpolynucleotide of claim
 1. 3. An isolated host cell comprising thevector of claim
 2. 4. A process for producing a polypeptide, comprisingculturing the host cell of claim 3 under conditions sufficient for theproduction of the polypeptide, wherein the polypeptide comprises SEQ IDNO:2.
 5. An isolated polynucleotide segment, comprising a nucleic acidsequence, wherein the nucleic acid sequence encodes a polypeptideconsisting of SEQ ID NO:2.
 6. A vector comprising the isolatedpolynucleotide segment of claim
 5. 7. An isolated host cell comprisingthe vector of claim
 6. 8. A process for producing the polypeptide,comprising culturing the host cell of claim 7 under conditionssufficient for the production of the polypeptide, wherein thepolypeptide consists of SEQ ID NO:2.
 9. An isolated polynucleotidesegment, comprising a nucleic acid sequence, wherein the nucleic acidsequence comprises SEQ ID NO:
 1. 10. A vector comprising the isolatedpolynucleotide segment of claim
 9. 11. An isolated host cell comprisingthe vector of claim
 10. 12. A process for producing the polypeptide,comprising culturing the host cell of claim 7 under conditionssufficient for the production of the polypeptide, wherein thepolypeptide comprises SEQ ID NO:2.